Optical density reader

Only free enzyme conjugate will bind to the coating. Addition of an enzyme substrate results in formation of a blue color, the intensity of which is inversely proportional to the amount of -lactam in the sample. Absence of -lactams in a sample results in all of the enzyme conjugate remaining unbound and available for binding to the immobilized -lactam. Presence of -lactams results in a portion of the enzyme conjugate being bound and unavailable to bind with the immobilized -lactam. The Delvo-X-Press test uses an optical density reader to compare the optical density of a standard set at a cutoff level of 5 ppb penicillin G, with that of each sample tube in total assay time of about 10 min. 

A confluent monolayer of Madin-Darby canine kidney (MDCK) cells was grown in 96-well plates. Serial tenfold dilutions in minimal essential medium were prepared from the aliquots of allantoic fluid taken from the irradiated specimen. These dilutions were applied to MDCK cells and incubated for 48 h at 36 °C in 5% C02. The cells were then washed two times for 5 min with phosphate buffered saline (PBS) and incubated for 1 h with 100 pi of 0.5 mg/ml solution of 3-(4,5-dimethyl-thiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT, ICN Biochemicals Inc., Aurora, Ohio). After lh, the colored deposit was dissolved in 100 pi DMSO, and optical density in the wells was measured on plate reader Victor 1420 (Perkin Elmer, Finland). Based on the data obtained, the infectious titer of the vims was determined as a decimal logarithm of reciprocal to the dilution of the specimen causing destruction of 50% of cells. The inhibiting action of irradiation was evaluated by decreasing the vims titer. 

Read the optical density (OD) on an ELISA plate reader Green ABTS reaction product and yellow ONPG reaction product may both be read at a wavelength of 406 nm... 

Ten milligrams of 2,2 -azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt is dissolved in 50 ml of substrate buffer containing 10 pi of 30% hydrogen peroxide. Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C, followed by visual plate reading on a 1+ to 4+ basis or at 540 pm if a microtiter plate reader is available. The optical density of the instrument is set to zero with the antigen control (well 12 in the first row). Any optical density in the antibody controls (bottom row) is subtracted from the corresponding test well above. 

There are many ways to estimate microbial growth. The simplest is visual inspection of colonies growing on agar plates, though this method is difficult to adapt for HTS. There is a well-known correlation between cell density and optical density, which can be exploited in a 96-well microtiter plate format (e.g., [43]). Measurement of the incorporation of radioactive nutrients is an excellent quantitative method, but has fallen from favor due to concerns about spills and contamination. Finally, both spectrophotometric and fluorimetric assays are conveniently adapted to HTS formats, and Alamar blue is only one example of the tools available for this purpose. As mentioned, we have even found it convenient to use simple visual inspection of Alamar blue plates to identify wells of interest. However, quantification obtainable with a microplate reader is attractive in many settings. 

Optical densities (OD) were read at 492 nm using an automated plate reader (Multiscan MS Labsystems, Helsinki, Finland) and cytokine levels calculated by interpolation from the standard curve (0.0-64 pg/ml). Data were corrected for protein concentration and the results expressed as pg of IL-1 f3 per mg of protein. 

If the optical density of a well is off-scale (i.e., >1.5 or 2.0, depending on the plate reader), dilute half the well s contents with an equal volume of water. The dynamic range of the assay can also be extended considerably by monitoring the color development in the amplification step kinetically rather than as an end point (8). This, however, requires a more sophisticated plate reader than is often available. A suitable instrument for kinetic reading is the Molecular Devices (Palo Alto, CA)Vmax. 

Analyses were done with a Molecular Devices microtiterplate reader interfaced to an IBM computer with Softmax software(Molecular Devices). Either the rate of color change (mOD/min) at 450 nm was recorded using the kinetic format or the final optical density (OD) was measured by difference of readings at 490 and 650 nm. 

Read the optical density at 490 nm of the reaction product by using a microplate reader. 

Measure the optical density D of the MTT-formazan solution after solubilization in the range of the wide absorption spectrum maximum (550-590 nm), for example, at 590 nm, using a microplate reader (e.g. Wallac Multilabel Counter measuring time 0.1 s). Use untransfected cells as a reference. Register the absorbance for one or several wells with a mixture of 100 pi MTT solution and 100 ml solubilization solution as a blank. 

Cytotoxicity test The L929 fibroblast cell line is the most widely used for cytotoxicity assessment 9-11. The cell metabolic activity was evaluated by succinc acid dehydrogenase (SDH) of mitochondria in living cell, which can deoxidized MTT salt into insoluble purple crystal. These crystals can be resolved by dimethyl sulfoxide (DMSO), and measured by the spectrophotometric microplate reader at 490 nm. To some extent, the optical density value (OD) of supernatant raised with the number of the living cell. Therefore, the measurement of MTT crystal OD can reflect the relative growth rate and the activity of cells, which can also leads to a reliable assessment to the cytotoxicity of the sample. 

The Soy Protein Residue Assay is a double-antibody (sandwich) ELISA using specific anti-soy tripsyn inhibitor and other soy protein antibodies coated onto microwells. After addition of the sample, the enzyme conjugate, and the TMB substrate, a positive reaction (indicating the presence of soy protein), produces a blue color. Addition of the stop solution ends the assay and turns blue to yellow. The result may be read visually (in the qualitative method) or with an ELISA reader (in the qualitative or quantitative method). Quantification can be obtained by mnning positive control standards (2.5-5-10-25 ppm) together with the samples. A standard curve is then plotted using the optical density (OD) values of the control standards (OD vs. concentration). 

Microtiter plates (MaxiSorp-immunoplates, NUNC A/S, Roskilde, Denmark) were coated overnight at 23°C with the cohesin test samples (200 pl/well, 270 nM of miniCipC c, wild-type or mutated Coh2CBD t). The plates were blocked for 2.5 h with blocking solution (300 pl/well 3% (w/v) bovine serum albumin in TrisNC buffer) and washed three times with TrisNC buffer (300 pl/well). The cohesin-dockerin interaction was initiated upon addition of dockerin samples (200 pl/well, 94 nM of XynDocA c or XynDocS t), and the plates were incubated for 2.5 h. After five washes, the bound dockerins were detected by means of the fused-xylanase activity substrate solution (240 pl/well 2.9 mM / -nitrophenyl p-D-cellobioside) was added followed by incubation at 60 C. Optical density was detOTnined at 420 nm on a VERSAmax microplate reader (Molecular Devices Corp., Sunnyvale CA). 

If a low volume pipet is to be calibrated in-house rather than being sent away to a certified calibration service that has suitable balances available, a method based on measurements of optical density of a dye solution, before and after dispensing an ahquot of water from the pipet, is used. This approach is called the Artel PCS2 system (www.artel-usa.com) a useful description of this system is readily available (Hemmings 2004a). This system makes use of a very accurate and precise dualwavelength UV-visible spectrophotometer that is readable to levels comparable to those of a microbalance. The method is based on principles aheady well established and used in microplate readers. 

Approximately 450 jjL serum-free DMEM and 50 pL CCK-8 solution were added to each sample, followed by incubation at 37 °C for 3 h to form water-soluble formazan. The supernatant was transferred into a 96-weU plate, and the optical density (OD) at 450 and 630mn was determined using a microplate reader (Multiskan MK3, Thermo Labsystems, Finland), with six parallel experiments of each sample used to assess cell viabihty. The adhered cells on films and scaffolds were fixed with 2.5% glutaralde-hyde (Alfa-Aesar) at 4°C for 12h. After thorough washing with PBS, cells adhered on films and scaffolds were dehydrated in an ethanol-graded series (50%, 60%, 70%, 80%, 90%, and 100%) for 15min each and allowed to dry via lyophilization to be used for SEM observations. 

We investigated the reactions on the MG-63 cell line and MCF-7 cell line in mixture with various amount of OA solution. DMEM-HG was used for cell culture as the basic media. The concentration of mixture was limited from 0% to 30%. DMEM-HG volume is fixed for minimizing its effect on cells. Total culture term was 14 days and MTT assay was performed on day 1, 3, 7, 10 and 14 throughout the experiment. For cell viability test, ELISA reader was used to measure the optical density (OD) at 595nm wavelength filter. 

Aliquots of 2QQhj of resulting MTT reaction were transferred in 96-well plate and optical density was measured at 595nm using ELISA reader (Enzyme-linked immunosorbent assay, Multiskan EX, Thermo Scientific Inc., USA) system. 

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